Developmental mechanisms underlying tooth patterning in continuously replacing osteichthyan dentitions

The dentition of osteichthyans presents an astonishing diversity with regard to the distribution of teeth in the oral cavity, tooth numbers, arrangements, shapes, and sizes. Taking examples from three unrelated teleosts--the most speciose group of osteichthyans--and from the literature, this study explores how the initial tooth pattern is set up, and how this relates to the establishment and maintenance (or modification) of the tooth replacement pattern. In teleosts, first-generation teeth (the very first teeth in ontogeny to develop at a particular locus) are commonly initiated in adjacent or in alternate (odd and even) positions. The mechanisms responsible for these divergent developmental patterns remain to be elucidated, in particular, whether they reflect a field or local type of control. However, patterns of adjacent or alternate tooth initiation, set up by the first-generation teeth, can easily turn into replacement patterns where new teeth are initiated simultaneously every second, or even every third position, by synchronizing the formation of new first-generation teeth to the formation of replacement teeth at older loci. Our observations suggest that, once established, the replacement pattern appears to be maintained, as a kind of "default" state. Variations and modifications in this pattern are nevertheless common and suggest that tooth replacement is under local control, exerted at the level of the initiation of replacement teeth. Further studies are needed to test the hypothesis that regular replacement patterns are more frequent in association with the plesiomorphic condition of extramedullary replacement (replacement on the surface of the dentigerous bone) and more rare in the derived condition of intramedullary replacement (replacement within the medullary cavity of the dentigerous bone).     

Human Tau Isoforms Assemble into Ribbon-like Fibrils That Display Polymorphic Structure and Stability

Fibrous aggregates of Tau protein are characteristic features of Alzheimer disease. We applied high resolution atomic force and EM microscopy to study fibrils assembled from different human Tau isoforms and domains. All fibrils reveal structural polymorphism; the “thin twisted” and “thin smooth” fibrils resemble flat ribbons (cross-section ∼10 × 15 nm) with diverse twist periodicities. “Thick fibrils” show periodicities of ∼65–70 nm and thicknesses of ∼9–18 nm such as routinely reported for “paired helical filaments” but structurally resemble heavily twisted ribbons. Therefore, thin and thick fibrils assembled from different human Tau isoforms challenge current structural models of paired helical filaments. Furthermore, all Tau fibrils reveal axial subperiodicities of ∼17–19 nm and, upon exposure to mechanical stress or hydrophobic surfaces, disassemble into uniform fragments that remain connected by thin thread-like structures (∼2 nm). This hydrophobically induced disassembly is inhibited at enhanced electrolyte concentrations, indicating that the fragments resemble structural building blocks and the fibril integrity depends largely on hydrophobic and electrostatic interactions. Because full-length Tau and repeat domain constructs assemble into fibrils of similar thickness, the “fuzzy coat” of Tau protein termini surrounding the fibril axis is nearly invisible for atomic force microscopy and EM, presumably because of its high flexibility.     

Imprecise excision of insertion element IS5 from the fliC gene contributes to flagellar diversity in Escherichia coli

Motile strains of Escherichia coli K12 carrying both a chromosomal fliC-H48 gene and a plasmid encoded fliC-H4 gene express both types of flagellins, which are coassembled into functional flagella. By using flagellar-H48-specific antiserum and a plasmid curing procedure, nonmotile mutants were found that carried an IS5 insertion in the chromosomal fliC-H48 gene. Motile revertants were isolated that showed deletions of the IS5 element together with sections of the fliC-H48 gene resulting in an altered flagellar serotype in these strains. As IS5 elements were found associated with 35 of 53 known H-types in wildtype E. coli strains, this insertion element might play a major role in serotype diversity.     

Robust translocation along a molecular monorail: the NS3 helicase from hepatitis C virus traverses unusually large disruptions in its track

The NS3 helicase is essential for replication of the hepatitis C virus. This multifunctional Superfamily 2 helicase protein unwinds nucleic acid duplexes in a stepwise, ATP-dependent manner. Although kinetic features of its mechanism are beginning to emerge, little is known about the physical determinants for NS3 translocation along a strand of nucleic acid. For example, it is not known whether NS3 can traverse covalent or physical discontinuities on the tracking strand. Here we provide evidence that NS3 translocates with a mechanism that is different from its well-studied relative, the Vaccinia helicase NPH-II. Like NPH-II, NS3 translocates along the loading strand (the strand bearing the 3'-overhang) and it fails to unwind substrates that contain nicks, or covalent discontinuities in the loading strand. However, unlike NPH-II, NS3 readily unwinds RNA duplexes that contain long stretches of polyglycol, which are moieties that bear no resemblance to nucleic acid. Whether located on the tracking strand, the top strand, or both, long polyglycol regions fail to disrupt the function of NS3. This suggests that NS3 does not require the continuous formation of specific contacts with the ribose-phosphate backbone as it translocates along an RNA duplex, which is an observation consistent with the large NS3 kinetic step size (18 base-pairs). Rather, once NS3 loads onto a substrate, the helicase can translocate along the loading strand of an RNA duplex like a monorail train following a track. Bumps in the track do not significantly disturb NS3 unwinding, but a break in the track de-rails the helicase.     

Single nucleotide polymorphism (SNP) genotyping as basis for developing a PCR-based marker highly diagnostic for potato varieties with high resistance to Globodera pallida pathotype Pa2/3

Globodera pallida is a parasitic root cyst nematode of potato, which causes reduction of crop yield and quality in infested fields. Field populations of G. pallida containing mixtures of pathotypes Pa2 and Pa3 (Pa2/3) are currently most relevant for potato cultivation in middle Europe. Genes for resistance to G. pallida have been introgressed into the cultivated potato gene pool from the wild, tuber bearing Solanum species S. spegazzinii and S. vernei. Selection of resistant genotypes in breeding programs is hampered by the fact that the phenotypic evaluation of resistance to G. pallida is time consuming, costly and often ambiguous. DNA-based markers diagnostic for resistance to G. pallida would facilitate the development of resistant varieties. A tetraploid F₁ hybrid family SR-Gpa segregating for quantitative resistance to G.␣pallida was developed and evaluated for resistance to G. pallida population ‘Chavornay’. Two subpopulations of 30 highly resistant and 30 susceptible individuals were selected and genotyped for 96 single nucleotide polymorphism (SNP) markers tagging 12 genomic regions on 10 potato chromosomes. Seven SNPs were found significantly linked to the nematode resistance, which were all located within a resistance ‘hotspot’ on potato chromosome V. A haplotype model for these seven SNPs was deduced from the SNP patterns observed in the SR-Gpa family. A PCR assay ‘HC’ was developed, which specifically detected the SNP haplotype c that was linked with high levels of nematode resistance. The HC marker was only found in accessions of S.␣vernei. Screening with the HC marker 34 potato varieties resistant to G. pallida pathotypes Pa2 and/or Pa3 and 22 susceptible varieties demonstrated that the HC marker was highly diagnostic for presence of high levels of resistance to G. pallida pathotype Pa2/Pa3.Amirali Sattarzadeh and Ute Achenbach contributed equally to the work     

The variability of ovum pick-up response and in vitro embryo production from monozygotic twin cows

Oocytes were recovered by ovum pick up (OPU) from nine pairs of monozygotic twin German Simmental cows. The hypothesis was that there is less variability between identical twins versus among non-related individuals in the variation in the recovery of oocytes by OPU and in the efficiency of in vitro embryo production. Estrous cycles were synchronized with two doses of cloprostenol, 11 days apart. Beginning 3-4 days after synchronized estrus, OPU was done twice weekly (every 3 or 4 days; total of 11 sessions). The influence of repeated OPU on estrous cyclicity was established by estrus detection, plasma progesterone concentrations, and ovarian ultrasonography. There were no differences among days of collection for the number and quality of cumulus oocyte-complexes (COCs), and rates of cleavage and blastocyst formation. A total of 1,661 COCs, including 657 (39.6%) good-quality COCs, were recovered. From 1,457 (87.7%) cultured COCs, 827 zygotes cleaved and 314 blastocysts were produced on Day 7. The total number of COCs and the blastocyst rates varied among pairs of monozygotic twins; within pairs, only slight differences were observed. In conclusion, recovery of COCs and production of embryos had substantially less variation within pairs of monozygotic twins than among non-related cattle.     

Mössbauer spectroscopy on respiratory complex I: the iron-sulfur cluster ensemble in the NADH-reduced enzyme is partially oxidized

In mitochondria, complex I (NADH:quinone oxidoreductase) couples electron transfer to proton translocation across an energy-transducing membrane. It contains a flavin mononucleotide to oxidize NADH, and an unusually long series of iron-sulfur (FeS) clusters that transfer the electrons to quinone. Understanding electron transfer in complex I requires spectroscopic and structural data to be combined to reveal the properties of individual clusters and of the ensemble. EPR studies on complex I from Bos taurus have established that five clusters (positions 1, 2, 3, 5, and 7 along the seven-cluster chain extending from the flavin) are (at least partially) reduced by NADH. The other three clusters, positions 4 and 6 plus a cluster on the other side of the flavin, are not observed in EPR spectra from the NADH-reduced enzyme: they may remain oxidized, have unusual or coupled spin states, or their EPR signals may be too fast relaxing. Here, we use M?ssbauer spectroscopy on (57)Fe-labeled complex I from the mitochondria of Yarrowia lipolytica to show that the cluster ensemble is only partially reduced in the NADH-reduced enzyme. The three EPR-silent clusters are oxidized, and only the terminal 4Fe cluster (position 7) is fully reduced. Together with the EPR analyses, our results reveal an alternating profile of higher and lower potential clusters between the two active sites in complex I; they are not consistent with the consensus picture of a set of isopotential clusters. The implications for intramolecular electron transfer along the extended chain of cofactors in complex I are discussed.     

A new mouse model for studying EGFR-dependent gastric polyps

Hyperactivation of the epidermal growth factor receptor (EGFR) in gastric cells due to excess of its ligand transforming growth factor-α (TGFA) is associated with hyperplastic lesions in Ménétrier's disease patients and in transgenic mice. Other EGFR ligands, however, have never been associated with stomach diseases. Here, we report that overexpression of the EGFR ligand betacellulin (BTC) results in a severe, age-dependent hyperplasia of foveolar epithelium. The stomach weight of affected mice reached up to 3g representing more than 10% of total body weight. The preexisting corpus mucosa was severely depleted, and both parietal and chief cells were replaced by proliferating foveolar epithelium. The lesions were more severe in male as compared to female transgenic mice, and partially regressed in the former after castration-mediated androgen removal. The gastric hyperplasia fully disappeared when BTC-tg mice were crossed into the Egfr(Wa5) background expressing a dominant-negative EGFR, indicating that the phenotype is EGFR-dependent. This is, to our knowledge, the first report of hyperplastic gastric lesions due to the overexpression of an EGFR ligand other than TGFA. BTC-tg mice are therefore a new, promising model for studying EGFR-dependent gastric polyps.     

Tau Protein Diffuses along the Microtubule Lattice

Current models for the intracellular transport of Tau protein suggest motor protein-dependent co-transport with microtubule fragments and diffusion of Tau in the cytoplasm, whereas Tau is believed to be stationary while bound to microtubules and in equilibrium with free diffusion in the cytosol. Observations that members of the microtubule-dependent kinesin family show Brownian motion along microtubules led us to hypothesize that diffusion along microtubules could also be relevant in the case of Tau. We used single-molecule total internal reflection fluorescence microscopy to probe for diffusion of individual fluorescently labeled Tau molecules along microtubules. This allowed us to avoid the problem that microtubule-dependent diffusion could be masked by excess of labeled Tau in solution that might occur in in vivo overexpression experiments. We found that approximately half of the individually detected Tau molecules moved bidirectionally along microtubules over distances up to several micrometers. Diffusion parameters such as diffusion coefficient, interaction time, and scanned microtubule length did not change with Tau concentration. Tau binding and diffusion along the microtubule lattice, however, were sensitive to ionic strength and pH and drastically reduced upon enzymatic removal of the negatively charged C termini of tubulin. We propose one-dimensional Tau diffusion guided by the microtubule lattice as one possible additional mechanism for Tau distribution. By such one-dimensional microtubule lattice diffusion, Tau could be guided to both microtubule ends, i.e. the sites where Tau is needed during microtubule polymerization, independently of directed motor-dependent transport. This could be important in conditions where active transport along microtubules might be compromised.     

Brief communication: a preliminary study on the influence of physical fruit traits on fruit handling and seed fate by white-handed Titi monkeys (Callicebus lugens)

Callicebus and the pitheciins are closely related; however, differences in their diets and dental morphology suggest that they differ in the use of mechanically protected food. We describe physical traits of fruits consumed by white-handed titi monkeys (Callicebus lugens) and determine their influence on fruit part selection and immediate seed fate after fruit handling. We tested two hypotheses about the effects of mechanical fruit traits on fruit part selection and seed fate: (1) fruits selected for seed consumption are harder than fruits selected for their fleshy parts and (2) consumed seeds are softer than seeds with other fates. In addition, we analyzed the influence of other physical fruit traits on fruit part selection and seed fate. C. lugens included 69 species in its diet, from which it mainly consumed their fleshy parts. It also consumed seeds, alone or with fleshy fruit parts, but most of them ended up close to parent trees after being dropped or spat out. The first hypothesis was supported while the second was rejected, indicating that C. lugens tends to rely on hard fruits for obtaining seeds, while seed hardness had no influence on fruit part selection and seed fate, contrasting with the pattern reported for Pithecia and Chiropotes in other studies. Ripeness was the most influential factor for fruit part and seed fate discrimination. Results suggest a tendency to sclerocarpic foraging in C. lugens when feeding on seeds.